CN113138272A

CN113138272A - Kit for detecting ubiquitin-conjugating enzyme antibody and use method thereof

Info

Publication number: CN113138272A
Application number: CN202110529869.5A
Authority: CN
Inventors: 刘煜; 钱莉; 朱昱晓
Original assignee: Sir Run Run Hospital Nanjing Medical University
Current assignee: Sir Run Run Hospital Nanjing Medical University
Priority date: 2021-05-14
Filing date: 2021-05-14
Publication date: 2021-07-20

Abstract

The invention discloses a kit for detecting ubiquitin-conjugating enzyme antibody and a using method thereof, belonging to the field of biomedicine. A kit for detecting antibodies to ubiquitin binding enzymes, comprising: a solution for detecting ubiquitin-binding enzyme antibodies; the solution comprises an active ester for labeling ubiquitin-conjugated enzyme antibodies and a non-radioactive solution. The active ester comprises active ester of thioruthenium derivative and active ester of biotin polyethylene glycol. The kit also comprises a 96-well plate, a streptavidin graphene plate, a 0.01mol/L PBS solution, a 5% BSA solution and a PBST solution. The streptavidin graphene plate is a streptomycin-labeled MSD96 pore plate. Compared with the prior art, the kit has the advantages of higher sensitivity and specificity, no radioactive substance, simple and convenient operation, capability of detecting the antibody amount in trace serum and good clinical application value.

Description

Kit for detecting ubiquitin-conjugating enzyme antibody and use method thereof

Technical Field

The invention relates to the field of biomedicine, in particular to a kit for detecting ubiquitin-conjugating enzyme antibody and a using method thereof.

Background

Classical T1DM is an antigen-specific T cell-mediated autoimmune disease that specifically damages islet beta cells, and islet autoantibodies play an important role in the prediction and diagnosis of T1 DM. The combined detection of four islet autoantibodies, GADA, IAA, IA-2A and ZnT8A, can predict 85% or more of T1DM patients or T1DM high risk groups. However, up to 15% of clinically diagnosed T1DM patients are negative for all currently known islet autoantibodies. Therefore, the discovery of new islet autoantibodies or the establishment of more accurate detection methods is a major approach to increasing autoantibody positive T1DM patients. Aiming at the defects of the prior art, provides a kit for detecting ubiquitin conjugated enzyme antibody and a using method thereof.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a kit for detecting ubiquitin conjugated enzyme antibodies.

The purpose of the invention can be realized by the following technical scheme:

a kit for detecting antibodies to ubiquitin binding enzymes, comprising: a solution for detecting ubiquitin-binding enzyme antibodies; the solution comprises active ester for labeling ubiquitin conjugated enzyme antigen protein, ubiquitin conjugated enzyme antigen protein and non-radioactive solution.

Further, the active ester comprises a thioruthenium derivative active ester and a biotin polyethylene glycol active ester.

Further, the kit also comprises a 96-well plate, a streptavidin graphene plate, a PBS solution, a BSA solution, a PBST solution and a Block-A solution.

Further, the PBS solution was 1 × PBS solution.

Further, the concentration of the BSA solution was 5%.

Further, the concentration of Block-A solution was 3%.

Further, the streptavidin graphene plate is a streptomycin-labeled MSD96 well plate.

The present invention also provides the use of a formulation for detecting ubiquitin-conjugating enzyme antibodies in a reagent for use in a method for detecting a subject in the presence of T1DM, said method comprising a method of use of said kit comprising the steps of:

marking recombinant antigen proteins of ubiquitin-conjugated enzyme by using active ester of thioruthenium derivative and biotin polyethylene glycol active ester respectively;

adding the marked antigen protein of the ubiquitin-conjugating enzyme into a solution of 5% BSA for dilution;

sequentially adding 16 mu l of 1 XPBS solution, 4 mu l of serum to be detected and diluted solution into each hole of a 96-hole plate, and uniformly stirring the mixed solution in the 96-hole plate;

placing the 96-hole plate in an oscillator, oscillating for 1h at a low speed, and storing the 96-hole plate in an environment at 4 ℃ for 6-12 h after oscillation is finished;

MSD96 well plates containing 150. mu.l/well of a 3% Block-A solution were stored at 4 ℃ for 6-12 hours; the stored MSD96 well plates were washed 3 times with 150 μ Ι PBST buffer;

transferring the mixed solution in the 96-well plate to an MSD96 pore plate, and placing the MSD96 pore plate filled with the mixed solution in a vibrator in a dark place for low-speed vibration for 1 h;

washing the MSD96 pore plate after the oscillation is finished for 3 times by using 150 mul of PBST buffer solution, and adding 150 mul/pore of Readingbuffer solution into the MSD96 pore plate after the washing is finished to count on an MSD counter;

and calculating the antibody index of the ubiquitin conjugated enzyme antibody according to the output result of the MSD counter.

The invention has the beneficial effects that:

the kit for detecting the ubiquitin-conjugating enzyme antibody can quickly determine the condition of a T1DM patient by detecting the ubiquitin-conjugating enzyme antibody; the kit has high sensitivity and specificity, no radioactive substances, simple and convenient operation, capability of detecting the antibody amount in trace serum and good clinical application value.

Drawings

The invention will be further described with reference to the accompanying drawings.

FIG. 1 is a graph of the positivity of UBE2L3-Ab of the present application in T1DM patients of various ages.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

193 patients with type 1 diabetes mellitus who are treated in outpatient clinics and wards of endocrinology department of a certain hospital are grouped according to the following standard: clinical diagnosis of T1DM and exclusion of patients with secondary diabetes, gestational diabetes and other autoimmune diseases according to 1999 World Health Organization (WHO) diagnostic criteria and typing recommendations (26); patient dependent exogenous insulin therapy; detecting at least one islet autoantibody, GADA, IAA, IA-2A or ZnT8, positive using a radioimmunocogand method; venous blood was collected from each subject on an empty stomach in the early morning for examination, and clinical data (name, sex, age, time of onset, course of disease, height, weight, glycated hemoglobin, C-peptide, ketosis, onset of hypoglycemia, insulin dosage, etc.) were recorded, and physical examinations were performed for height, weight, etc.

The specific experimental method comprises the following steps: using electrochemiluminescence, i.e., labeling recombinant ubiquitin-conjugated enzyme (UBE2L3) antigen proteins (as can be queried from https:// www.ncbi.nlm.nih.gov/gene/7332) with a thioruthenium derivative active ester (Sulfo-tag) and Biotin polyethylene glycol active ester (Biotin), respectively, binding the antibodies in the serum sample with the labeled antigens:

16. mu.l of 1 XPBS solution, 4. mu.l of serum to be tested, UBE2L3 antigen which is diluted with 5% BSA and has been linked to Sulfo-tag and Biotin are added to a 96-well plate in sequence, mixed uniformly and placed in an oscillator for 1h at room temperature and low speed, and then the 96-well plate is placed in a refrigerator at 4 ℃ for 6-12 h.

A streptavidin-labeled MSD96 well plate was pre-loaded with 150. mu.l/well of a 3% Block-A solution and the MSD96 well plate was placed in a refrigerator at 4 ℃ for 6-12 hours. After the end of the set, the MSD96 well plate was washed 3 times with 150. mu.l PBST buffer.

Sequentially sucking 30 μ l of the mixed solution from each well of the 96-well plate and transferring the mixed solution to an MSD 96-well plate;

after the MSD96 well plate was shaken in the dark at room temperature for 1 hour, washed again with PBST buffer for 3 times, and then added with 150. mu.l of Readingbuffer solution to count on the MSD counter;

the antibody index was calculated by the formula: the antibody index is (serum to be tested-negative quality control)/(positive quality control-negative quality control).

The experimental results are as follows:

1. determination of the Positive cut-off value of UBE2L3-Ab for the test population

UBE2L3-Ab of 124 healthy people was detected by ECL, the median of UBE2L3-Ab index was-0.065 (-0.036, 0.087), 99% of percentile was taken, and the positive cut-off value of UBE2L3-Ab was 0.037.

2. The positive rate of the T1DM group is obviously higher than that of the healthy control group

UBE2L3-Ab was positive in 19 of 193 patients with incipient T1DM with a positive rate of 9.84%, in 3 of 282 healthy controls with a positive rate of 1.06%, and in T1DM group, the difference was significantly higher than that in the healthy controls, with statistical significance (P < 0.005).

3. Positive rate of antibody of different age groups

The T1DM population was divided into four subgroups by age at diagnosis, with groups <18, 18-29, 30-39 and >40 years of age, respectively. The positive rate of the UBE2L3 antibody was 10.96% (8/73) in the 0-18 year old group, 16.67% (9/54) in the 19-29 year old group, 2.94% (1/34) in the 29-39 year old group, and 3.13% (1/32) in the 40 year old group. The positive rate of UBE2L3-Ab peaked at age 18-29 and then declined, with significant differences between >40 age subgroups compared to the other three subgroups (mean P < 0.05; as shown in figure 1).

And (4) experimental conclusion: the prevalence rate of UBE2L3-Ab in T1DM patients is significantly higher than that of healthy control groups, and the positive rates of the UBE2L3-Ab in various subgroups of different onset ages are significantly different, so that the UBE2L3-Ab can be used as a condition detection index of T1DM patients.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed.

Claims

1. A kit for detecting antibodies to ubiquitin-conjugating enzymes, comprising: a solution for detecting ubiquitin-binding enzyme antibodies; the solution comprises active ester for labeling ubiquitin conjugated enzyme antigen protein, ubiquitin conjugated enzyme antigen protein and non-radioactive solution.

2. The kit for detecting ubiquitin conjugated enzyme antibody of claim 1, wherein the active ester comprises thioruthenium derivative active ester and biotin polyethylene glycol active ester.

3. The kit for detecting ubiquitin-conjugating enzyme antibody according to claim 2, wherein the kit further comprises a 96-well plate, a streptavidin graphene plate, a PBS solution, a BSA solution, a PBST solution and a Block-A solution.

4. The kit for detecting ubiquitin conjugated enzyme antibody of claim 3, wherein the PBS solution is 1 XPBS solution.

5. The kit for detecting ubiquitin conjugated enzyme antibody of claim 3, wherein the concentration of BSA solution is 5%.

6. The kit for detecting ubiquitin-conjugating enzyme antibody according to claim 3, wherein the concentration of Block-A solution is 3%.

7. The kit for detecting ubiquitin-conjugating enzyme antibody of claim 3, wherein the streptavidin graphene plate is a streptomycin labeled MSD96 well plate.

8. Use of a preparation for detecting ubiquitin-conjugating enzyme antibodies in a reagent for use in a method for detecting a subject in the presence of T1DM, the method comprising:

washing the MSD96 pore plate after the oscillation is finished for 3 times by using 150 mul of PBST buffer solution, and adding 150 mul/pore Reading buffer solution into the MSD96 pore plate after the washing is finished to count on an MSD counter;